PROJECT SUMMARY/ABSTRACT Cancer is an intimate part of us, recruiting a complex array of endogenous cellular processes to drive its fitness. Cancer initiation and progression is frequently the result of coordinated dysregulation of multiple signaling pathways by key oncogenes that strategically coopt the cancer signaling network. These oncogenes rarely work in isolation but instead form intricate higher-order complexes and participate in multifaceted networks of protein- protein interactions. One of the most commonly mutated proteins across all of human cancers is the phosphoinositide 3-kinase (PI3K) oncogene, a lipid kinase that can exploit diverse cellular programs to drive disease, including increased proliferation, survival, motility, cell growth and metabolic activity. Broad inhibition of PI3K is known to generate systemic toxicities, especially metabolic, which limit its clinical development. However, targeting PI3K interacting proteins may enable a safer alternative, by interrupting PI3K oncogenic activity while minimizing metabolic dysregulation. The long-term goal of this proposal is to deepen and refine our understanding of oncogene regulation of cancer signaling networks using systematic genetic, proteomic, and mathematical modeling approaches. The overall objective of this proposal is to identify the role of PI3K interacting proteins in modulating PI3K activity and recruitment of downstream cellular processes and to use this understanding to identify alternative therapeutic targets. This objective will be reached by testing the central hypothesis that PI3K interacting proteins, or downstream signaling pathways, can be modulated to tune PI3K activity and specificity, with the potential to simultaneously reduce malignancy and systemic toxicity. To test this hypothesis, the following three aims will be pursued. (Aim 1) Reveal Regulation of PI3K Activity and Cancer Phenotypes by PIK3CA Interacting Proteins. This aim will use CRISPR/Cas9 gene knockout technology to systematically delete and overexpress genes corresponding to PIK3CA interacting proteins and use live cell microscopy, combined with biochemical measurements, to assess the resulting impact on cell proliferation, survival, growth, motility, and metabolism. (Aim 2) Elucidate Proteomic Exploitation by PI3K as Mediated by PIK3CA Interacting Proteins. Here, PIK3CA interactors that preferentially bind the common H1047R mutant, as well as hits identified from Aim 1, will be subject to global proteomics and phosphoproteomics profiling to identify signaling pathways and biological processes regulated by each PIK3CA interacting protein. (Aim 3) Delineate Mechanisms of PI3K-mediated Manipulation of Pro-Cancer Signaling. Here, a novel computational framework will be developed by uniting data-driven network propagation techniques with mechanistic ordinary differential equation (ODE) modeling to delineate mechanistic pathways linking each PIK3CA interacting protein to its downstream effect. Successful completion of the proposed research will greatly enhance our mechanistic understanding of oncogene regulation in cancer. This will be a significant contribution as it will reveal novel therapeutic strategies to fight cancer while minimizing systemic toxicities.